The present invention relates to the detection of pathogens and toxins, particularly to a highly flexible liquid array that utilizes optically encoded microbeads as the templates for biological assays, and more particularly to a micro-immunoassay (handheld) system wherein target biological samples are optically labeled and captured on microbeads, which are in turn captured on an ordered array or disposable capture substrate and optically read.
The most commonly employed portable pathogen detection is strip-type tests, such as those used in handheld glucose diagnostics or the Joint Biological Point Detection System (JBPDS), a system used for detection of biowarfare agents. These tests are held or ‘smart ticket’ assay, and are currently the smallest embodiment of a viable pathogen detection technology. In the JBPDS, for example, a membrane strip is printed with three lines: a mobile line of colored latex particles coated with an antibody to the bioagent being detected, a fixed line of a second antibody to the same bioagent, and a fixed line of antibody directed to the antibody on blue latex particles. To perform an assay, a liquid sample is added to the device that hydrates the latex spheres (which are located in the sample well). If the targeted bioagent is present, a complex is formed between the latex sphere and bioagent. This complex wicks through the strip and is captured by the fixed line of antibody to the bioagent forming a visible line of color. A line will also appear at the next fixed line due to capture of free latex spheres. Thus a negative assay will only have a single line at the control line and a positive assay will have two lines.
The JBPDS obtains multiplex capability by delivering multiple “tickets” (printed membrane strips) to the assay by means of a mechanical carousel. Currently, nine different “tickets”, each sensitive to a different bioagent, share the sample and perform the analysis with fluidic automation and photonic inspection of the test lines. This technology represents a credible solution for military use since the number of target pathogens is limited. For civilian use, however, the scaling of the device to 30 or more pathogens is quite problematic. The carousel becomes increasingly complicated and large, while dividing the sample between the different assays creates an unacceptable reduction in sensitivity.
The ‘DNA Chip’ on the other hand, is an emerging technology based on nucleic acid identification of pathogens that has a proven multiplex capability. The chip has its origin in the publication “Light-directed spatially addressable parallel chemical synthesis,” Fodor et al, (1991) Science, 251, p. 767-773. Here a format was proposed that allowed for a very dense packing of chemical probe compounds on a silicon substrate. Affymetrix Corp., Santa Clara, Calif. is developing the most promising version of this technology, see U.S. Pat. No. 6,045,996 issued Apr. 4, 2000 to Cronin et. al. By combining solid phase chemical synthesis with modern photolithographic fabrication techniques, Affymetrix Corp. has been able to assemble an array of 65,000 individual and unique nucleic acid probes in an area of several square centimeters. Each probe region consists of thousands of identical molecules in an area or ‘patch’ 50 microns square. This resulting silicon surface can analyze a complex solution for specific DNA fragments by forming hybridization with its nucleic acid component that has been labeled with a fluorescent dye. In the DNA chip's basic application, replicates of the DNA being analyzed are produced, fractionated and flourescently labeled. They are then exposed to the chip and allowed a period of time to find a complementary sequence on the chip. If this occurs hybridization occurs at the sight or patch containing the complementary DNA sequence. This results in a flourescently labeled region that is read by a raster scanning laser beam and suitable collection optics that preserve spatial resolution.
As powerful as the chip technology is, it does have some significant drawbacks. A single chip design from concept to product can cost as much as $400K. In addition, once its design is determined it cannot be altered. The instrumentation to read the chip is very large and optically fragile. There is a lengthy incubation time to allow for the hybridization of the target. In applications such as bioforensics or proteonics, where new information is constantly being made available, one is committed to a series of costly chip developments to keep the analysis current. Finally, individual chips can be reused only a few times, so this technology is impractical for application requiring the analysis of hundred to thousands of samples.
A sensor array for the measurement and identification of multiple analytes in solution has been developed by J. T. McDevitt et al, University of Texas, as published in International Publication No. WO00/04372 on 27 Jan. 2000, based on U.S. application Ser. No. 09/207,248 filed 7 Apr. 1999. This involves a system for rapid characterization of multi-analyte fluids and, in one embodiment, includes a light source, a sensor array, and a detector. The sensor array is formed from a supporting member into which a plurality of cavities may be formed. A series of chemically sensitive particles, beads, or microspheres are positioned within the cavities. The particles may be configured to produce a signal when a receptor coupled to the particle interacts with the analyte. Using patterned recognition techniques, the analytes within a multi-analyte fluid may be characterized.
A multiplex detection system using a Liquid Array is a more flexible and cost-effective format than either the JBPDS or the DNA chips, described above. By use of the Liquid Array, additional assays can be added simply by addition of different color bead sets. Up to 100-plex assay can now be performed using a 10×10 array of microbead sets developed by Luminex Corporation, Austin, Tex. under U.S. Pat. No. 6,057,107 issued May 2, 2000 to J. R. Fulton. Each microbead is individually doped with two fluorescent dyes (orange and red) as indicated in FIG. 1, wherein a liquid array shows the absolute intensity of the two dyes (orange and red) as indicated by legend and arrows and which provides a method to uniquely identify each microbead set.
Given the existing Luminex Corp. bead set, two different types of multiplex analysis can now occur. The first involves multiplexed detection of different biomarkers in the sample, as seen in FIG. 2 wherein each bead color is used to identify a specific bioagent assay. In this approach, a sample is added to a collection of microbeads. Each color or microbeads contains a capture assay that is specific for a given bioagent. Fluorescent labels are then added to identify the presence of each agent on the bound bead.
In the second type of multiplexed analysis, using the bead set, different microbead colors are used to identify the sample rather than the bioagent. Here, all the microbeads are labeled with the same bioagent assay, with each person assigned a different color, see FIG. 3. Since all the samples can be run in a highly parallel fashion, victims potentially exposed to a pathogen could quickly be screened.
Luminex Corp. developed the Liquid Array concept of FIGS. 1-3 to be used in conjunction with a benchtop flow cytometer. Each optically encoded and flourescently labeled microbead is individually counted for the fraction of a second it passes through the detection system, creating the need for a complex fluidics and optoelectronics package. The Luminex Corp. flow cytometer, therefore, is well suited for laboratory analysis but is neither inexpensive nor compact enough to be used in field or chair-side measurements.
The present invention which utilizes the liquid array approach of Luminex Corp., as described above, involves a method for constructing a portable pathogen detection system that accomplished on-site multiple detection of targets in biological samples. In the system of the invention, a highly flexibly Liquid Array utilizes optically encoded microbeads as the templates for biological assays. The system of this invention basically contains microbead specific reagents, incubation/mixing chambers, a microbead capture array substrate, and an optical measurement and decoding system. Target biological samples are optically labeled and captured on the microbeads, which are in turn captured on an ordered array and optically read.
This invention combines the probability of the smart ticket (JBPDS), the scalability of the DNA chips, and flexibility of the Luminex Corp. flex cytometer to create a powerful multiplex detection platform as set forth in Table 1 shown comparison of MIDS to “Chip” and “Liquid Arrays.” This invention intentionally moves away from a laboratory type instrument, where extensive training and elaborate biochemical protocols are necessary. The goal was to approach the simplicity and probability of a JBPDS strip test without compromising the flexibility, sensitivity, and multiplicity of the liquid array paradigm. Like the strip test, the entire sample preparation module is intended to be disposable, with microbead imaging and detection performed on a separate reader device. The use of a disposable, in addition to being a good diagnostic business model, eliminates cross sample contamination and greatly simplified the fluidics.
TABLE 1ChipLiquid ArrayMIDSFlexibilityFixedBeads readilyBeads readilytemplateexchanged in setexchanged in setInstrument˜$250K$30K$10KCostAssay Cost$400K setup$30K set-up plus$40K set-up plusplus $200$1 per multiplex$2 per multiplex assayper assayassaySpeedHoursMinutesMinutesTargetNucleic acidDNA, toxin,DNA, toxin, spore,spore, virus,virus, bacteriabacteriaPortabilityBenchtopBenchtopHandheldRobustnessControlledControlledField EnvironmentEnvironmentEnvironment